CN104982225A - Novel male silkworm cordyceps culturing process and method for culturing silkworm cordyceps parent species - Google Patents
Novel male silkworm cordyceps culturing process and method for culturing silkworm cordyceps parent species Download PDFInfo
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- CN104982225A CN104982225A CN201510383370.2A CN201510383370A CN104982225A CN 104982225 A CN104982225 A CN 104982225A CN 201510383370 A CN201510383370 A CN 201510383370A CN 104982225 A CN104982225 A CN 104982225A
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Abstract
The invention relates to a novel male silkworm cordyceps culturing process comprising the following steps: (1) a silkworm cordyceps strain is selected; (2) a silkworm cordyceps parent species is cultured, and is prepared into a bacterium liquid; (3) male silkworm cordyceps culturing is carried out. A silkworm cordyceps parent species culturing method suitable for the novel male silkworm cordyceps culturing process comprises the following steps: (1) separation is carried out, wherein mature silkworm cordyceps fruiting bodies are separated and cultured in petri dishes; (2) transplanting is carried out, wherein silkworm cordyceps strains growing well in the peri dishes are transplanted into slant media in test tubes and are cultures; (3) rejuvenation is carried out, wherein the silkworm cordyceps strains on the slant media in the test tubes are prepared into the bacterium liquid; the bacterium liquid is injected into silkworm bodies, such that strain activity is enhanced; (4) mature silkworm cordyceps fruiting bodies grown on the silkworm bodies are subjected to separation and transplanting operations again according to the above steps (1)-(3). According to the invention, silkworm cordyceps is artificially cultured with male silkworm larvae as a host. Compared to a method for culturing cordyceps with silkworm pupa, labor is saved, and cost is low.
Description
Technical field
The present invention relates to Chinese caterpillar fungus and cultivate field, be specifically related to a kind of novel male silkworm Chinese caterpillar fungus cultivating technique.
Background technology
Cordyceps sinensis is a kind of famous and precious Chinese herbal medicine, there is nourishing the lung and the kidney, hemostasis and phlegm, anti-oxidant, anti-inflammatory, the multiple remarkable medical active such as anticancer, modern medical analysis is thought, cordycepin, Cordyceps sinensis polysaccharide, adenosine, cordycepic acid are the main active of Chinese caterpillar fungus, and cordycepin has the multiple pharmacological effect such as antitumor, anti-ageing, antibacterial, antiviral, immunological regulation, improvement metabolism, scavenging free radicals; Cordyceps sinensis polysaccharide can improve immune function of human body, increasing leukocyte, is clinically used for the treatment of malignant tumour; Adenosine has coronary artery dilating blood vessel, increases the effect of CBF; Cordycepic acid can prevent and treat cerebral thrombus, cerebral hemorrhage, miocardial infarction, long-term decline.These active components of Cordyceps sinensis all have good potential applicability in clinical practice, and the market demand is very big.The height of cordycepin, Cordyceps sinensis polysaccharide, adenosine, cordycepic acid content is one of main standard weighing Chinese caterpillar fungus quality, it is generally acknowledged that the medical value of the Chinese caterpillar fungus that cordycepin, Cordyceps sinensis polysaccharide, adenosine, cordycepic acid content are high is high.
But due to by the restriction of ecotope and the impact of excessively excavating, this rare resources is petered out, and natural cs resource can not be met the need of market far away.Adopt the alternative that factory culturing artificial aweto becomes best.The Chinese caterpillar fungus of artificial culture is approved as new resource food by health ministry.
In the market artificial cultivation Chinese caterpillar fungus mainly utilize rice (wheat) and silkworm chrysalis to be host cultivates out, wherein Cordyceps militaris studies artificial aweto the most widely.Compared with Cordyceps sinensis, Cordyceps militaris is tight to the requirement of host, and primary medicinal component is close with Cordyceps sinensis, therefore carry out Cordyceps militaris artificial cultivation can Cordyceps Resources be in short supply opens up an effective way for solution.But still there is following subject matter in current Chinese caterpillar fungus artificial breeding technique: (1) microorganism resource is degenerated, and active component content is unstable; (2) culture conditions harshness is difficult to accomplish scale production; (3) artificial aweto product shortcoming deep development.
Show through early-stage Study of the present invention, inoculate Cordyceps Militaris using silkworm as host and cultivate the silkworm Chinese caterpillar fungus obtained, the content of its active component cordycepin, Cordyceps sinensis polysaccharide, adenosine, cordycepic acid is all higher than Cordyceps militaris and natural cordyceps, and silkworm Chinese caterpillar fungus form than Cordyceps militaris closer to natural cs, therefore silkworm Chinese caterpillar fungus has very wide value of exploiting and utilizing.But, the development and utilization of silkworm Chinese caterpillar fungus also exists bacterial classification instability, culture conditions harshness, be difficult to problems demand solutions such as accomplishing scale production.
Summary of the invention
In order to solve the above-mentioned problems in the prior art, the present invention proposes a kind of process utilizing male silkworm inoculation Cordceps militaris bacterium to produce high nutrient functional product, specific as follows:
A kind of novel male silkworm Chinese caterpillar fungus cultivating technique, comprises step:
1) silkworm cordyceps species is selected;
2) cultivate silkworm Chinese caterpillar fungus mother to plant, and make bacterium liquid;
3) male silkworm Chinese caterpillar fungus is cultivated, and step comprises:
3.1) the large dormancy opening male silkworm of 2-5 days silkworm alive is selected to do host;
3.2) sterilization prepares;
3.3) adopt the method for injection to inoculate, injection volume is every bar silkworm 2 ± 0.2 microlitre;
3.4) postvaccinal host is cultivated in an aseptic environment:
3.4.1) the dead rigid phase:
A, cleaning:
Infect about 5 days, have part silkworm to harden, they are detected, deposits separately, dry-run protection; Infect 9 ~ 10 days, silkworm foreign material are with it removed to remaining sclerosis silkworm, deposits separately, dry-run protection;
The silkworm use water hardened is eluriated clean, then rinses to without muddy water with water purification, then rinse;
B, to dry:
Shakeout by the silkworm body of clean sclerosis, shady and cool local air-dry, degree of drying feels with hand not to be felt wet and slippery, is as the criterion without obviously water stain;
3.4.2) bacteria developing period (10th ~ 16 days from inoculation starts)
The silkworm of drying is divided in box, must not stack; After mounted box completes, cover lid is put in dark surrounds and is carried out dark protection 5 ~ 6 days; Wherein the initial temperature of environment is 17 ~ 18 DEG C, then heats 1 DEG C every 1 ~ 2 day, until temperature reaches 22 ~ 23 DEG C, when silkworm body covers with the white bacterium of sesame size;
3.4.3) colour-change period (17th ~ 21 days from inoculation starts)
A, photosensitive:
To silkworm body whole day, make full use of scattering and carry out illumination, must the time-division shelters from heat or light at noon between illumination period;
Between illumination period, must with fluorescent lamp light source as a supplement when light is inadequate; Should be noted that the mycelial growth situation of observing silkworm body, first long white hypha, then grows grass;
B, moisturizing:
Note observing the moisture situation in box, lid there to be the tiny globule be advisable, as without the globule, moisturizing be carried out; Moisturizing adopts sterile water to spray;
3.4.4) the grass phase is gone out
A, growth: (22nd ~ 30 days from inoculation starts)
The temperature in vegetative period controls at 22 ~ 23 DEG C;
B, ripe harvesting:
Being 30 ~ 40 days from being inoculated into results, being advisable not grow ascospore; The temperature of picking period controls at 22 ~ 23 DEG C.
Described step 1) in, it is ergot Zoopagales that silkworm Chinese caterpillar fungus mother plants the bacterial classification selected, Clavicipitaceae, Cordyceps, silkworm Cordyceps Militaris.
Described step 3) in, select male silkworm silkworm alive to do host; Male silkworm silkworm alive is the cultivation stage silkworm, adopts fluorescence processing method, is separated by male silkworm seed, cultivates respectively with female silkworm seed;
Adopt to add the antibiotic mode of food to resist the infection of virus and other miscellaneous bacteria to the male silkworm silkworm that lives;
Antibiotic: allocation ratio 1g selects Florfenicol to be dissolved in 1.5 ~ 2.5 kilograms of jin water, sprays 20 kilograms of mulberry leaf, adds the male silkworm of food; If second day has fragmentary dead silkworm, antibiotic concentration doubles.
Described step 3.1) large dormancy opening refer to the 4th dormancy phase terminate to become fifth instar larva after (casting off a skin for the 4th time after terminating), and come into play and eat mulberry leaf; Do not need to anaesthetize, directly the silkworm Cordyceps Militaris liquid configured is injected male silkworm silkworm body.
Described step 3.2) in, the bacterium liquid of employing needs operate under the gnotobasis of aseptic operating platform to configure into, only containing in steps 1) the silkworm Cordyceps Militaris one bacterium that obtains; Before injector to inject, high pressure-temperature was sterilized, and had often injected needle tubing, carried out once sterilizing with alcohol.
Described step 3.3) in, flat for needle point gesture, at silkworm drosal part the 3rd link place, is inserted the intersegmental membrane skin of silkworm body, is thrust the degree of depth to guarantee that silkworm can not be as the criterion in death by injection position; Press wound with have gentle hands when extracting, prevent body fluid from flowing out.
The method that the cultivation silkworm Chinese caterpillar fungus mother being applicable to described novel male silkworm Chinese caterpillar fungus cultivating technique plants, comprises step:
1) be separated: the silkworm cordyceps militaris sporocarp of maturation is carried out lock out operation, cultivates in culture dish;
What medium adopted is solid culture medium, and filling a prescription is:
1,200g potato adds the filtered fluid that poach obtains for 20 minutes;
2, glucose 20g;
3, agar 20g;
4, peptone 2g;
5, potassium dihydrogen phosphate 1g;
6, magnesium sulfate 0.3g;
7, constant volume is added water in 1000ml,
The operating procedure that silkworm cordyceps species is separated is: the silkworm cordyceps militaris sporocarp 1, selecting eugonic, longer thicker bacterial classification more is separated
2, cut fruit body with tweezers and little scissors and put into aseptic culture dish
3, first carry out disinfection by the alcohol-pickled fruit body sheared off of 70%-75%
4,0.1% mercuric chloride soaking disinfection is used again, the recyclable recycling of mercuric chloride
5, to have sterilized rear aseptic water washing three times, disinfectant is not remained
6, a bit of fruit body sheared off is separated from centre again, be divided into three little fourths
7, put three little fourths in a culture dish, place the bacterial classification cut in angular distribution
8, cover culture dish, write bacterium numbering and date, seal loading freshness protection package with sealing strip
9, culture dish is put into biochemical cultivation case together with freshness protection package to cultivate, temperature is set as 23 DEG C, and humidity is because be so do not control in the culture dish of sealing.General 2 day time, mycelia can grow, and within 4-5 days, white hypha grows up to, and in little circular flora, diameter is normal at about 5mm.
2) transplant: the operating procedure of transplanting: 1, grafting device sterilized on alcolhol burner, sterilization distance is longer, and hand-held handle also will carry out disinfection.
2, remove the sealing strip on culture dish, the good flora of growth selection is transplanted
3, open the sterile grafting device of culture dish scrapes on the good flora of growth selected, and grafting device just brings bacterial classification
4, grafting device is stretched in vitro, complete in " Z " font uniform application from inside to outside on slant medium
5, after having transplanted, test tube mouth and test tube plug are crossed on alcolhol burner, disappear and put in poison.
6, by test tube plug beyond the Great Wall, outside newspaper is wrapped and is tied with line.Write the date of bacterium numbering and transplanting exactly
7, put into biochemical cultivation case to cultivate, about about 5-7 days invisible spectro slant mediums cover with mycelia
8, approximately after about 10 days, mycelia is comparatively abundant with regard to length
9, put into 5 DEG C of refrigerators, refrigerate about 20-25 days, then could be used for rejuvenation inoculation and use.
By identical for the medium growing good silkworm cordyceps species in culture dish and be transplanted in the slant medium (medium and step 1) of test tube) on cultivate;
3) rejuvenation: the silkworm cordyceps species on slant medium in test tube is mixed with bacterium liquid and is expelled to silkworm body, injection volume is 2 ± 0.2 microlitres/bar silkworm, impels bacterial activity to strengthen;
The preparation method of bacterium liquid is:
1) mortar is carried out disinfection sterilizing;
2) spoon and handle thereof are carried out disinfection;
3) spoon handle is stretched in vitro bacterial classification is scraped totally;
4) bacterial classification is scraped in mortar grind, then add sterile water, then grind;
5) getting filter paper is placed on bacterium liquid liquid level, draws bacterium liquid, be placed in aseptic bottle with dropper;
6) last, the broken bacterial classification in test tube, in test tube, rinses, draws after grinding again by pouring;
7)---grinding---absorption that adds water repeats, and floccule should filter clean by period;
8) finally sterile water is supplemented in aseptic bottle according to concentration requirement;
9) the bacterium liquid drawn in aseptic bottle is inserted in centrifuge tube, and bacterium liquid completes and can use.
The storage temperature of bacterium liquid is 4 ± 0.5 DEG C; The concentration of bacterium liquid is: the quality of silkworm cordyceps species accounts for 9 ~ 12%.
4) by the silkworm cordyceps militaris sporocarp of the maturation that silkworm silkworm body grows according to above-mentioned steps 1) ~ 3) re-start separation and graft procedure.
Described silkworm body is the live body of male silkworm; Require to be (cast off a skin for the 4th time after terminating) after the 4th dormancy phase terminating to become fifth instar larva in vegetative period of silkworm body, and come into play and eat mulberry leaf.
The present invention is with male silkworm larva for host's artificial cultivation silkworm Chinese caterpillar fungus, and Chinese caterpillar fungus is short compared with silkworm chrysalis man-hours requirement, to save labour, cost low to utilize male silkworm larva to cultivate.Early-stage Study shows; the active component cordycepic acid of silkworm Chinese caterpillar fungus and the content of cordycepin are all higher than Cordyceps militaris and natural cordyceps; and silkworm Chinese caterpillar fungus form than Cordyceps militaris closer to natural cs; therefore silkworm Chinese caterpillar fungus has more wide value of exploiting and utilizing, and the large-scale production of silkworm Chinese caterpillar fungus can obtain significant Social and economic benef@.
Embodiment
The technical program is further illustrated as follows below in conjunction with embodiment:
1, the selecting of silkworm cordyceps species:
The worm grass product of high-quality at least should ensure 2 points: the active constituent contents such as the pure of bacterial classification and sufficiently high cordycepin, Cordyceps sinensis polysaccharide, adenosine, cordycepic acid.The bacterial classification that the technical program is selected is ergot Zoopagales, Clavicipitaceae, Cordyceps, silkworm Cordyceps Militaris, adopts this bacterial classification as mother's kind.
2, the cultivation of silkworm Chinese caterpillar fungus mother kind:
The cultural method that silkworm Chinese caterpillar fungus mother plants, comprises step:
1) be separated: the silkworm cordyceps militaris sporocarp of maturation is carried out lock out operation, cultivates in culture dish;
What medium adopted is solid culture medium, and filling a prescription is:
1,200g potato adds the filtered fluid that poach obtains for 20 minutes;
2, glucose 20g;
3, agar 20g;
4, peptone 2g;
5, potassium dihydrogen phosphate 1g;
6, magnesium sulfate 0.3g;
7, constant volume is added water in 1000ml,
The operating procedure that silkworm cordyceps species is separated is: the silkworm cordyceps militaris sporocarp 1, selecting eugonic, longer thicker bacterial classification more is separated
2, cut fruit body with tweezers and little scissors and put into aseptic culture dish
3, first carry out disinfection by the alcohol-pickled fruit body sheared off of 70%-75%
4,0.1% mercuric chloride soaking disinfection is used again, the recyclable recycling of mercuric chloride
5, to have sterilized rear aseptic water washing three times, disinfectant is not remained
6, a bit of fruit body sheared off is separated from centre again, be divided into three little fourths
7, put three little fourths in a culture dish, place the bacterial classification cut in angular distribution
8, cover culture dish, write bacterium numbering and date, seal loading freshness protection package with sealing strip
9, culture dish is put into biochemical cultivation case together with freshness protection package to cultivate, temperature is set as 23 DEG C, and humidity is because be so do not control in the culture dish of sealing.General 2 day time, mycelia can grow, and within 4-5 days, white hypha grows up to, and in little circular flora, diameter is normal at about 5mm.
2) transplant: the operating procedure of transplanting: 1, grafting device sterilized on alcolhol burner, sterilization distance is longer, and hand-held handle also will carry out disinfection.
2, remove the sealing strip on culture dish, the good flora of growth selection is transplanted
3, open the sterile grafting device of culture dish scrapes on the good flora of growth selected, and grafting device just brings bacterial classification
4, grafting device is stretched in vitro, complete in " Z " font uniform application from inside to outside on slant medium
5, after having transplanted, test tube mouth and test tube plug are crossed on alcolhol burner, disappear and put in poison.
6, by test tube plug beyond the Great Wall, outside newspaper is wrapped and is tied with line.Write the date of bacterium numbering and transplanting exactly
7, put into biochemical cultivation case to cultivate, about about 5-7 days invisible spectro slant mediums cover with mycelia
8, approximately after about 10 days, mycelia is comparatively abundant with regard to length
9, put into 5 DEG C of refrigerators, refrigerate about 20-25 days, then could be used for rejuvenation inoculation and use.
By identical for the medium growing good silkworm cordyceps species in culture dish and be transplanted in the slant medium (medium and step 1) of test tube) on cultivate;
3) rejuvenation: the silkworm cordyceps species on slant medium in test tube is mixed with bacterium liquid and is expelled to silkworm body, injection volume is 2 ± 0.2 microlitres/bar silkworm, impels bacterial activity to strengthen;
The preparation method of bacterium liquid is:
1) mortar is carried out disinfection sterilizing;
2) spoon and handle thereof are carried out disinfection;
3) spoon handle is stretched in vitro bacterial classification is scraped totally;
4) bacterial classification is scraped in mortar grind, then add sterile water, then grind;
5) getting filter paper is placed on bacterium liquid liquid level, draws bacterium liquid, be placed in aseptic bottle with dropper;
6) last, the broken bacterial classification in test tube, in test tube, rinses, draws after grinding again by pouring;
7)---grinding---absorption that adds water repeats, and floccule should filter clean by period;
8) finally sterile water is supplemented in aseptic bottle according to concentration requirement;
9) the bacterium liquid drawn in aseptic bottle is inserted in centrifuge tube, and bacterium liquid completes and can use.
The storage temperature of bacterium liquid is 4 ± 0.5 DEG C; The concentration of bacterium liquid is: the quality of silkworm cordyceps species accounts for 9 ~ 12%.
4) by the silkworm cordyceps militaris sporocarp of the maturation that silkworm silkworm body grows according to above-mentioned steps 1) ~ 3) re-start separation and graft procedure.
Described silkworm body is the live body of male silkworm; Require to be (cast off a skin for the 4th time after terminating) after the 4th dormancy phase terminating to become fifth instar larva the vegetative period of silkworm body, come into play and eat mulberry leaf.
The quality of mother culture media to bacterial classification plays an important role.After testing, this method is adopted to cultivate the mother's kind obtained, the content 13.2% that the content of its cordycepin is 1.2mg/g, the content of Cordyceps sinensis polysaccharide is 3.94g/100g, the content of adenosine is 85mg/100g, cordycepic acid.
3, silkworm Chinese caterpillar fungus is cultivated:
Select the large dormancy opening 2-5 days male silkworms of work to do host, now young silkworm body is big and fleshy greatly on the one hand, and gained silkworm Chinese caterpillar fungus is also large; Another aspect silkworm now does not also weave silk.The silk gland protein of percentage of liveweight about 40% becomes the nutrition of Cordyceps Militaris, and nutrition and the active component of gained silkworm Chinese caterpillar fungus are the highest.But large dormancy opening 2-5 days silkworm premunitions alive are very strong, are difficult to inoculation Cordyceps Militaris.Therefore the inoculating tool and the inoculation method that improve silkworm Chinese caterpillar fungus is necessary.Set up ecological standardization workshop of cultivating silkworm Chinese caterpillar fungus in addition, set up a set of asepticize method of operating, thus realize the large-scale production of silkworm Chinese caterpillar fungus.
Nutritive value and the medium of Chinese caterpillar fungus are closely related, market is generally at present: 1. use rice, Testa Tritici, agar etc. to be that medium produces Chinese caterpillar fungus, can only the use of dining table, nutritive value is lower, 2. silkworm chrysalis high-temperature sterilization inoculation, output is high, but after killing pupa alive, because of silk-fibroin high temperature meeting sex change more than 60 degree, enzyme also loses activity.3. silkworm chrysalis of living is inoculated, and because the complete silk of curley waste could become pupa, such protein content is less.
The advantage of this method: 1. with large dormancy opening 2-5 days silkworm inoculation alive, protein in Chinese caterpillar fungus, organized enzyme are higher.2. this product adopts the male silkworm inoculation (male and female silkworm adopts fluorescent technique process screening) in 2-5 days of large dormancy opening, the Chinese caterpillar fungus produced, according to test: cordycepin content reaches 2.3%, higher than wild cordyceps 8 times, other benefit materials content all exceedes or is equivalent to wild cordyceps, have the effect of tonifying both YIN and YANG, especially man's tonifying liver, function for tonifying kidney are better.
Active ingredient and the natural cs of this silkworm Chinese caterpillar fungus are basically identical, and what have is even better than natural cs.And silkworm raises maximum insects in the world, utilizing silkworm larva to cultivate Chinese caterpillar fungus has abundant available resources.Now inoculate as parasitism inoculation Cordyceps Militaris, to finding best training method with the opening 2-5 days silkworms alive that sleep greatly.With inoculate Cordyceps Militaris with silkworm chrysalis and compare, required time is short, and save a large amount of manpower and materials, cost is low, reduces and blindly gathers natural cs resource, can obtain more considerable economic benefit, thus recycles developing frontier for Silkworm resource.
The important and difficult issues of the invention
(1) degenerate problem of silkworm cordyceps species
Because silkworm Chinese caterpillar fungus requires with general edible mushroom different to growth substrate and ecotope, and the long-term silkworm cordyceps species adopting vegetative propagation and repeatedly tube, its female gene of planting easily makes a variation, and show as out careless deformity and even do not go out grass, seed output and quality is all unstable.
Therefore, and technical solution of the present invention research and find out a kind of method effectively preventing cordyceps species from degenerating, successfully solve the problem that silkworm Cordyceps Militaris is easily degenerated, significantly enhance vigor and the appeal of silkworm Cordyceps Militaris, inoculation rate reaches 100%, and host's survival rate can reach more than 90%)
(2) during batch production easily there is septicemia in the silkworm of inoculation
Find in practice, easily there is septicemia in the silkworm inoculated during batch production, how to take measures to address this problem from each side such as Spawn incubation, inoculation and environmental Kuznets Curves, the present invention innovates to some extent in the establishment of the further bionical cultivation facility condition of silkworm Chinese caterpillar fungus, both reached in country's medicine, food production management and required the condition of sterile working, met again the growth of silkworm Chinese caterpillar fungus to temperature, light, the requirement such as to wet.Specific solving methods: (one) selects the silkworm inoculation alive in 2-5 days of large dormancy opening to be parasitic inoculation Cordyceps Militaris, and now silkworm egg stalwartness is not easily fallen ill; (2) select suitable disinfectant to carry out disinfection to silkworm body before injection inoculation, kill the various pathogenic microorganisms be attached on silkworm body as far as possible, particularly cause septicemic cemia malignant bacteria.(silkworm body is not sterilized, because sterilization is difficult to thoroughly, and easily infects.What adopt is carry out spraying antibiotic then to the method for silkworm feeding to mulberry leaf, strengthens silkworm to the resistivity of disease)
Postvaccinal host is cultivated in an aseptic environment:
3.4.1) the dead rigid phase:
Cleaning:
To infection about 5 days, there is part silkworm to harden, detected, be placed in plastic casing, dry-run protection.Deng sclerosis silkworm stiff degree better time (general 9 ~ 10 days) remove sclerosis silkworm foreign material with it, be placed in plastic casing protection.The silkworm hardened is picked up and eluriates clean with water, then rinse without muddy water with tap, then rinse, be advisable not have impurity.Should be noted that to pick out as far as possible and infect silkworm body that is bad and that be bacterial contamination.
Dry:
The silkworm body of clean sclerosis is shakeout, dries, can not get sun, shady and cool local air-dry.(time is not strict with, with hand feel do not feel wet and slippery, without obviously water stain just can, can not be too dry).Should be noted that to pick out as far as possible and infect silkworm body that is bad and that be bacterial contamination.
3.4.2) bacteria developing period
Dark protection:
The silkworm of drying is divided in box, can squeeze, but not stack, in order to avoid affect hyphal development.The shelf that after mounted box completes, cover lid is put in dark room carries out dark and protects (general 5-6 days); initial temperature be 17-18 DEG C (can not higher than 20 DEG C); then 1 DEG C was heated every 1-2 days; until temperature reaches 22-23 DEG C for going out careless optimal temperature (this process generally continues about 5-6 days), when silkworm body covers with the white bacterium of sesame size.Should be noted that to pick out as far as possible and infect silkworm body that is bad and that be bacterial contamination.
3.4.3) colour-change period
This, mycelia was transformed by Bai Xianghuang in period, and grew tiny fruit body.
1, photosensitive:
This period requires high to light, needs abundant illumination.Can scattered light be made full use of, but can not sunshine direct projection, must the time-division shelters from heat or light at noon.At this moment need 24 hours photosensitive, must with fluorescent lamp light source (best fluorescent lamp opens for 24 hours, can suitably close for a moment when daytime is bright and clear) as a supplement when light is inadequate.Should be noted that the mycelial growth situation of observing in box, first long white hypha (a little as mouldy), then grows grass.Should be noted that to pick out as far as possible and infect silkworm body that is bad and that be bacterial contamination.
2, moisturizing:
Note observing the moisture situation in box, lid there to be the tiny globule be advisable, as without the globule, moisturizing be carried out.Moisturizing adopts sterile water, directly can buy or use with pouring in bottle to spray after boiling water cooling.
3.4.4) the grass phase is gone out
This, fruit body started to grow in period
1, grow:
The temperature of growth controls at 22-23 DEG C, and temperature is too low then grows slowly, and temperature is too high then aging.Moisture can suitably supplement.This stage notes the growing state observing fruit body.
2, ripe harvesting:
From being inoculated into results general 30-40 days (being generally as the criterion with 35 days), being no more than 45 days, being advisable not grow ascospore.Grow thecasporous excessively old.Period, temperature stabilization controlled at 22-23 DEG C.
The preparation method of bacterium liquid is:
Utensil prepares: aseptic bottle, Nian Portland, dropper, spoon, bacterial classification, filter paper, sterile water, ethanol for disinfection, beaker, centrifuge tube, alcolhol burner etc.Sterilization: aseptic bottle, Nian Portland, spoon, sterile water, centrifuge tube, beaker etc. carry out high pressure-temperature sterilizing.
Configuration bacterial classification suspension:
1) mortar is poured into 75% ethanol for disinfection to carry out disinfection, then with aseptic water washing once
2) spoon is sterilized on alcolhol burner, long handle also will be crossed on flame.
3) spoon handle is stretched in vitro bacterial classification is scraped totally.
4) bacterial classification is scraped in mortar, grind gently, add 10-20ml sterile water, then grind
5) getting filter paper is placed on bacterium liquid liquid level, draws bacterium liquid, be placed in aseptic bottle with dropper
6) when etc. bacterium liquid is less, the broken bacterial classification in test tube, in test tube, rinses by pouring, draws gently after grinding again
7) add water-grind-draw and can repeat, note floccule to filter totally
8) finally see in aseptic bottle the bacterium liquid having how many capacity, add water to about 225ml.
9) the bacterium liquid drawn in aseptic bottle is inserted in centrifuge tube, and bacterium liquid completes and can use.
The storage temperature of bacterium liquid is 4 DEG C and is advisable; The concentration of bacterium liquid is: the quality of silkworm cordyceps species accounts for about 10%.
" Cordyceps sinensis " and " silkworm Chinese caterpillar fungus " testing result adopting the technology of the present invention to produce are in table 1
Table 1: silkworm Chinese caterpillar fungus is compared with Cordyceps sinensis active component
| Composition | The content of silkworm Chinese caterpillar fungus | The content of Cordyceps sinensis | Detection method |
| Cordycepin | 1.2mg/g | 0.001mg/g | GF245 plate layer chromatography method |
| Cordycepic acid | 13.2％ | 6.94％ | TLC analytical method |
| Adenosine | 85mg/100g | 39.6mg/100g | GF245 plate layer chromatography method |
| Cordyceps sinensis polysaccharide | 3.94g/100g | 2.5g/100g | Fehling reagent titration |
| Ergosterol | 2.28mg/g | 1.22mg/g | TLC analytical method |
| SOD | 78u/g | 30u/g | Phosphorus benzenetriol Autoxidation Method |
| Selenium | 0.6ppm | 0.42ppm | Atomic absorption spectrography (AAS) |
Claims (10)
1. a novel male silkworm Chinese caterpillar fungus cultivating technique, is characterized in that comprising step:
1) silkworm cordyceps species is selected;
2) cultivate silkworm Chinese caterpillar fungus mother to plant, and make bacterium liquid;
3) male silkworm Chinese caterpillar fungus is cultivated, and step comprises:
3.1) the large dormancy opening male silkworm of 2-5 days silkworm alive is selected to do host;
3.2) sterilization prepares;
3.3) adopt the method for injection to inoculate, injection volume is every bar silkworm 2 ± 0.2 microlitre;
3.4) postvaccinal host is cultivated in an aseptic environment:
3.4.1) the dead rigid phase:
A, cleaning:
Infect about 5 days, have part silkworm to harden, they are detected, deposits separately, dry-run protection; Infect 9 ~ 10 days, silkworm foreign material are with it removed to remaining sclerosis silkworm, deposits separately, dry-run protection;
The silkworm use water hardened is eluriated clean, then rinses to without muddy water with water purification, then rinse;
B, to dry:
Shakeout by the silkworm body of clean sclerosis, shady and cool local air-dry, degree of drying feels with hand not to be felt wet and slippery, is as the criterion without obviously water stain;
3.4.2) bacteria developing period
The silkworm of drying is divided in box, must not stack; After mounted box completes, cover lid is put in dark surrounds and is carried out dark protection 5 ~ 6 days; Wherein the initial temperature of environment is 17 ~ 18 DEG C, then heats 1 DEG C every 1 ~ 2 day, until temperature reaches 22 ~ 23 DEG C, when silkworm body covers with the white bacterium of sesame size;
3.4.3) colour-change period
A, photosensitive:
To silkworm body whole day, make full use of scattering and carry out illumination, must the time-division shelters from heat or light at noon between illumination period;
Between illumination period, must with fluorescent lamp light source as a supplement when light is inadequate; Should be noted that the mycelial growth situation of observing silkworm body, first long white hypha, then grows grass;
B, moisturizing:
Note observing the moisture situation in box, lid there to be the tiny globule be advisable, as without the globule, moisturizing be carried out; Moisturizing adopts sterile water to spray;
3.4.4) the grass phase is gone out
A, growth:
The temperature in vegetative period controls at 22 ~ 23 DEG C;
B, ripe harvesting:
Being 30 ~ 40 days from being inoculated into results, being advisable not grow ascospore; The temperature of picking period controls at 22 ~ 23 DEG C.
2., according to its novel male silkworm Chinese caterpillar fungus cultivating technique according to claim 1, it is characterized in that described step 1) in, it is ergot Zoopagales that silkworm Chinese caterpillar fungus mother plants the bacterial classification selected, Clavicipitaceae, Cordyceps, silkworm Cordyceps Militaris.
3., according to its novel male silkworm Chinese caterpillar fungus cultivating technique according to claim 1, it is characterized in that described step 3) in, select male silkworm silkworm alive to do host; Male silkworm silkworm alive is the cultivation stage silkworm, adopts fluorescence processing method, is separated by male silkworm seed, cultivates respectively with female silkworm seed;
Adopt to add the antibiotic mode of food to resist the infection of virus and other miscellaneous bacteria to the male silkworm silkworm that lives;
Antibiotic: allocation ratio 1g selects Florfenicol to be dissolved in 1.5 ~ 2.5 kilograms of jin water, sprays 20 kilograms of mulberry leaf, adds the male silkworm of food; If second day has fragmentary dead silkworm, antibiotic concentration doubles.
4., according to its novel male silkworm Chinese caterpillar fungus cultivating technique according to claim 1, it is characterized in that described step 3.1) after large dormancy opening refers to that the 4th the dormancy phase of silkworm terminate, come into play and eat mulberry leaf, reach fifth instar larva; Do not need to anaesthetize, directly the silkworm Cordyceps Militaris liquid configured is injected male silkworm silkworm body.
5. according to its novel male silkworm Chinese caterpillar fungus cultivating technique according to claim 1, it is characterized in that described step 3.2) in, the bacterium liquid adopted all need operate and configures under the gnotobasis of aseptic operating platform, only containing in steps 1) obtain and not containing the silkworm Cordyceps Militaris one bacterium of other miscellaneous bacteria; Before injector to inject, high pressure-temperature was sterilized, and had often injected needle tubing, carried out once sterilizing with alcohol.
6. according to its novel male silkworm Chinese caterpillar fungus cultivating technique according to claim 1, it is characterized in that described step 3.3) in, flat for needle point gesture, at silkworm drosal part the 3rd link place, is inserted the intersegmental membrane skin of silkworm body, is thrust the degree of depth to guarantee that silkworm can not be as the criterion in death by injection position; Press wound with have gentle hands when extracting, prevent body fluid from flowing out.
7. the method that the cultivation silkworm Chinese caterpillar fungus mother being applicable to the arbitrary described novel male silkworm Chinese caterpillar fungus cultivating technique of claim 1 ~ 6 plants, is characterized in that comprising step:
1) be separated: the silkworm cordyceps militaris sporocarp of maturation is carried out lock out operation, cultivates in culture dish;
2) transplant: cultivate on the slant medium growing good silkworm cordyceps species in culture dish and be transplanted to test tube;
3) rejuvenation: the silkworm cordyceps species on slant medium in test tube is mixed with bacterium liquid and is expelled to silkworm body, injection volume is 2 ± 0.2 microlitres/bar silkworm, impels bacterial activity to strengthen;
4) by the silkworm cordyceps militaris sporocarp of the maturation that silkworm silkworm body grows according to above-mentioned steps 1) ~ 3) re-start separation and graft procedure.
8. the method for cultivation silkworm Chinese caterpillar fungus mother kind according to claim 7, is characterized in that described silkworm body is the live body of male silkworm; The vegetative period of silkworm body requires to be, after the 4th dormancy phase terminating to become fifth instar larva, come into play and eat mulberry leaf.
9. the method for cultivation silkworm Chinese caterpillar fungus mother kind according to claim 7, is characterized in that step 1) in,
A) operating procedure that silkworm cordyceps species is separated is:
A-1) select eugonic silkworm cordyceps militaris sporocarp to be separated;
A-2) cut fruit body with tweezers and little scissors and put into aseptic culture dish;
A-3) first carry out disinfection by the alcohol-pickled fruit body sheared off of 70%-75%;
A-4) 0.1% mercuric chloride soaking disinfection is used again, the recyclable recycling of mercuric chloride;
A-5) to have sterilized rear aseptic water washing three times, disinfectant is not remained;
A-6) a bit of fruit body sheared off is separated from centre again, be divided into three little fourths;
A-7) put three little fourths in a culture dish, place the bacterial classification cut in angular distribution;
A-8) cover culture dish, write bacterium numbering and date, seal loading freshness protection package with sealing strip;
A-9) culture dish is put into biochemical cultivation case together with freshness protection package to cultivate, temperature is set as 23 DEG C, and humidity is because be so do not control in the culture dish of sealing.General 2 day time, mycelia can grow, and within 4-5 days, white hypha grows up to, and in little circular flora, diameter is normal at about 5mm;
What the medium b) in culture dish adopted is solid culture medium, and filling a prescription is:
B-1) 200g potato adds the filtered fluid that poach obtains for 20 minutes;
B-2) glucose 20g;
B-3) agar 20g;
B-4) peptone 2g;
B-5) potassium dihydrogen phosphate 1g;
B-6) magnesium sulfate 0.3g;
B-7) constant volume is added water in 1000ml;
Step 2) in, the operating procedure of transplanting is:
2-1) grafting device sterilized on alcolhol burner, hand-held handle also will carry out disinfection.
2-2) remove the sealing strip on culture dish, the flora that growth selection is good is transplanted;
2-3) open the sterile grafting device of culture dish scrapes on the good flora of growth selected, and grafting device just brings bacterial classification;
2-4) grafting device is stretched in vitro, complete in " Z " font uniform application from inside to outside on slant medium;
After 2-5) having transplanted, test tube mouth and test tube plug are crossed on alcolhol burner, sterilization;
2-6) by test tube plug beyond the Great Wall, outside newspaper is wrapped and is tied with line; Write the date of bacterium numbering and transplanting exactly;
2-7) put into biochemical cultivation case to cultivate, about 5 ~ 7 days invisible spectro slant mediums cover with mycelia:
2-8) after 9 ~ 11 days, mycelia just looks abundant:
2-9) put into 5 DEG C of refrigerators, refrigerate 20 ~ 25 days, then could be used for rejuvenation inoculation and use;
Medium and the step 1 of this step) medium identical;
Step 3) in, the preparation method of bacterium liquid is:
1) mortar is carried out disinfection sterilizing;
2) spoon and handle thereof are carried out disinfection;
3) spoon handle is stretched in vitro bacterial classification is scraped totally;
4) bacterial classification is scraped in mortar grind, then add sterile water, then grind;
5) getting filter paper is placed on bacterium liquid liquid level, draws bacterium liquid, be placed in aseptic bottle with dropper;
6) last, the broken bacterial classification in test tube, in test tube, rinses, draws after grinding again by pouring;
7)---grinding---absorption that adds water repeats, and floccule should filter clean by period;
8) finally sterile water is supplemented in aseptic bottle according to concentration requirement;
9) the bacterium liquid drawn in aseptic bottle is inserted in centrifuge tube, and bacterium liquid completes and can use.
10. the method for cultivation silkworm Chinese caterpillar fungus mother kind according to claim 7, is characterized in that step 3) in, the storage temperature of bacterium liquid is 4 ± 0.5 DEG C; The concentration of bacterium liquid is: the quality of silkworm cordyceps species accounts for 9 ~ 12%.
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